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REVIEW ARTICLE
Wilson Parawira
Department of Applied Biology, Kigali Institute of Science and Technology (KIST), Avenue de I Armee, B.P. 3900 Kigali,
Rwanda
Abstract
Biogas technology provides an alternative source of energy to fossil fuels in many parts of the world. Using local
resources such as agricultural crop remains, municipal solid wastes, market wastes and animal waste, energy (biogas),
and manure are derived by anaerobic digestion. The hydrolysis process, where the complex insoluble organic
materials are hydrolysed by extracellular enzymes, is a rate-limiting step for anaerobic digestion of high-solid organic
solid wastes. Biomass pretreatment and hydrolysis are areas in need of drastic improvement for economic production
of biogas from complex organic matter such as lignocellulosic material and sewage sludge. Despite development of
pretreatment techniques, sugar release from complex biomass still remains an expensive and slow step, perhaps the
most critical in the overall process. This paper gives an updated review of the biotechnological advances to improve
biogas production by microbial enzymatic hydrolysis of different complex organic matter for converting them into
fermentable structures. A number of authors have reported significant improvement in biogas production when
crude and commercial enzymes are used in the pretreatment of complex organic matter. There have been studies on
the improvement of biogas production from lignocellulolytic materials, one of the largest and renewable sources of
energy on earth, after pretreatment with cellulases and cellulase-producing microorganisms. Lipids (characterised as
oil, grease, fat, and free long chain fatty acids, LCFA) are a major organic compound in wastewater generated from the
food processing industries and have been considered very difficult to convert into biogas. Improved methane yield
has been reported in the literature when these lipid-rich wastewaters are pretreated with lipases and lipase-producing
microorganisms. The enzymatic treatment of mixed sludge by added enzymes prior to anaerobic digestion has been
shown to result in improved degradation of the sludge and an increase in methane production. Strategies for enzyme
dosing to enhance anaerobic digestion of the different complex organic rich materials have been investigated.
This review also highlights the various challenges and opportunities that exist to improve enzymatic hydrolysis of
complex organic matter for biogas production. The arguments in favor of enzymes to pretreat complex biomass
are compelling. The high cost of commercial enzyme production, however, still limits application of enzymatic
hydrolysis in full-scale biogas production plants, although production of low-cost enzymes and genetic engineering
are addressing this issue.
Keywords: Enzymes, hydrolysis, complex organic matter, anaerobic digestion, improved biogas production
Introduction
Address for Correspondence: Parawira Wilson, Department of Applied Biology, Kigali Institute of Science and Technology (KIST), Avenue de
I Armee, B.P. 3900 Kigali, Rwanda. Email: parawiradr@yahoo.co.uk
(Received 19 November 2010; revised 17 May 2011; accepted 06 June 2011)
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the cellulose and hemicelluloses more accessible for
further enzymatic hydrolysis. Microorganisms capable of
converting polysaccharides to monosaccharides include
cellulase-producing Trichoderma reesei and Trichoderma
viride. Srilatha etal. (1995) obtained a 33% methane yield
increase from orange processing waste by treatment with
selected fungi strains. Similarly, Taherzadeh and Karimi
(2008) reported improved enzymatic hydrolysis (94%
sugar recovery) when treating office paper with selected
aerobic bacteria. An edible mushroom, Pleurotus sajorcaju, which is cultivated on a variety of agricultural
residues, possesses the capacity to degrade cellulose,
hemicelluloses and lignin components (Bisaria et al.,
1983). Biogas can then be produced from the degraded
agricultural wastes. Muller and Trosch (1986) reported
twice as much biogas yield from fungus (Pleurotus sp.
florida) treated straw than from untreated straw. They
had grown twenty two basidiomycetes, mostly white
rot fungi on wheat straw and found that this oyster
mushroom showed fastest delignification of the straw.
Their results showed that myco-straw can be better
hydrolysed and converted to biogas in comparison to
untreated straw. After biological lignin removal the straw
cellulose was better accessible for anaerobic digestion.
The procedure involving microbial delignification and
biogas production, offers the possibility of utilizing and
removing the waste wheat straw in a completely biological way. The useful products from this process are
mushroom in the first step and methane in the second
step. Geeta et al. (1994) found that sugarcane bagasse
pretreated with Phanerochaete chrysosporium for 3 weeks
under ambient temperature conditions produced higher
biogas compared with cattle excreta. Phanerochaete
chrysosporium is the most investigated fungus for lignin
degradation. Akao et al. (1992) reported enhanced
anaerobic digestion of citrus peels with an enzyme
solution from Aspergillus sp. A-1. The enzyme solution
was reported to have cellulase and pectinase activities
that allowed the anaerobic digester to be operated at a
higher organic loading rate. Biological ensilage additives
with hetero- and homo-fermentative activity (Silasil
energy) as well as enzymes (Sil-all 4 x4) or bacteria
and yeasts (Microferm) effectively increased the biogas
production per organic dry matter of maize (11.8, 10.1,
and 14.7%, respectively) after 7 weeks of pretreatment
(Vervaeren etal., 2010). However, treatment rate is very
low in most biological pretreatment processes. At present
the process of decomposing cellulose and other complex
molecules into simple sugars and other compounds
relies on naturally occurring bacteria. The efficiency of
cellulose breakdown could be improved by using better
strains, particularly for cellulolysis, the limiting step of
the process. Guedon et al. (2002) demonstrated that
fermentation of cellulose, the most abundant and renewable polymer on earth, can be greatly improved by using
a genetically engineered Clostridium cellulolyticum
with improved cellulolytic properties. Bagi et al. (2007)
demonstrated an increase of biogas production by about
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2006; Leal et al., 2006). Mendes et al. (2006) investigated the effect of enzymatic hydrolysis pretreatment
of lipids-rich dairy wastewater on anaerobic digestion.
A low-cost commercially available lipase preparation
from an animal source was used to perform enzymatic
hydrolysis pretreatment of lipid-rich wastewater from
dairy industry which resulted in increased levels of biogas production and higher organic (COD) removal. The
pretreatment was optimized for a 12h hydrolysis time
enabling high biogas formation and organic removal.
Promising results in relation to the biogas formation were
also found when the hydrolysis and biodegradation steps
were carried out simultaneously. Another application
of low-cost enzymes for pretreatment of effluents from
poultry slaughterhouses was reported by Valladao etal.
(2007). A pool of hydrolases with 21.4U g1 lipase activity was produced through solid-state fermentation of the
fungus Penicillium restrictum in waste from the Orbignya
oleifera (babassu) oil processing industry. Enzymatic
hydrolysis and anaerobic biodegradability tests were
conducted on poultry slaughterhouse effluents with
increased oil and grease contents (1501200mg l1) and
solid enzymatic pool concentration (0.11.0%) w/v).
Enhanced anaerobic treatment efficiency relative to raw
effluent (COD removal efficiency of 85% versus 53% and
biogas production of 175ml versus 37ml after 4 days) was
achieved when a 0.1% concentration of enzymatic pool
was used in the pretreatment stage with 1200mg oil and
grease l1. Cammarota et al. (2001) reported that enzymatic pretreatment using an enzymatic cocktail of lipases
resulted in improvement of anaerobic degradation in an
UASB reactor of dairy wastewater containing elevated fat
levels compared with untreated wastewater. The dairy
wastewater was pre-treated with 0.1% (w/v) of fermented
babassu cake containing Penicillium restrictum lipases
obtained from solid state fermentation. The application
of low cost lipases as a pretreatment to hydrolyze and
dissolve lipids may improve the anaerobic degradation
of wastewaters rich in lipids, enhancing the production
of biogas. With slaughterhouse wastewater, pancreatic
lipase PL 250 (424h) reduced the average particle size
by 60% and increased lipid hydrolysis by 40% during 24h
(Mendes etal., 2006). Luste etal. (2009) investigated the
use of a biological product containing hydrolytic enzymes
during solubilization of organic material, i.e., hydrolysis
and methane production potentials on different byproducts from meat-processing plants. The biological
product Liquid Certizyme 5TM increased soluble COD
of digestive tract and drumsieve waste the most as compared to untreated material, (62% and 96%, respectively).
However, methane production potential was decreased
compared to untreated materials apparently due to
inhibition by hydrolysis products such as VFA from
acidification of solubilized products. Masse etal. (2001a)
observed that 35% of the natural fats in slaughterhouse
effluents hydrolysed to free LCFA when treated with pancreatic lipase 250 (PL-250), increasing the rate of removal
in an anaerobic reactor operating in batch conditions.
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Lab-scale
Mixtures of enzymes
Pilot-scale
Full-scale
Mixtures of enzymes
Two glycosidic enzymes
Lab-scale
Lab-scale
Mixture of enzymes
(cellulase& pronase E)
Cellulase, protease, lipase
Amylase, hemicellulase
Effects
Improved solubilisation of
organicmatter (2040%)
Reducedvitality
Increased methane production
(60%)
Increased methane yield (60%)
Increased methane yield (71%)
Increase methane
production(20%)
Improved dewatering
properties
Enhanced dewaterability
(basedoncapillary suction
hydrolytic enzymes, time,
solidcontent of final product,
filtrate turbidity)
Reduction of total
solids (80%)
Improved solid reduction
andsettling of solids
Reference
Wawrzynczyk
etal., 2003
Davidsson
etal., 2007
Wawrzynczyk, 2007
Ayol, 2005
by Lagerkvist and Chen (1993) in their study of a twostep anaerobic degradation of municipal solid waste by
enzyme addition.
In continuous pilot-scale experiments, Davidsson
etal. (2007) reported a higher methane yield (0.398 Nm3
CH4/kg VSin) achieved when enzymes were added directly
into the digester together with fresh sludge compared
with 0.366 Nm3 CH4/kg VSin when the enzyme was added
with digested sludge via a return pipe. A comparison
between methane potentials and methane yields showed
that 9799% of the methane potential could be realised
in methane yield when enzymes were used. For the
untreated sludge 75% of the potential was realised in
continuous digestion.
Wawrzynczyk (2007) reported improved biogas production and dewatering properties in a continuously
operating mesophilic full-scale wastewater treatment
plant during a six month period after addition of
two technical grade glycosidic enzymes supplied by
Novozymes A/S, Denmark. The dosage was 2.5kg of each
enzyme solution per tonne feed dry solids to the digestion chamber. The dosage point was the heat exchanger
system which ran every fourth hour for 30 to 40 minutes.
Preceding laboratory tests to determine the types of
enzymes and the dosage optimum had showed that the
two selected enzymes resulted in a better solubilisation of the sludge than proteases and lipases. The gas
production from enzyme treated sludge increased by 10
to 20% in comparison to that from the reference digester
without enzyme. These results with studies done using
laboratory-, pilot- and full-scale reactors demonstrate
the potential to enhance biogas production from sewage
sludge using enzymes. Possible strategies for enzyme
dosing to enhance anaerobic digestion of sewage sludge
for biogas production have been attempted and the
results are encouraging.
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Table 2. Summary of advantages and disadvantages of using enzymes to hydrolyse organic rich waste.
Advantages
Organic rich wastes hydrolysis using enzymes is done under mild, and non-corrosive and quick physical-chemical operating conditions
Enzymes are specific and effective unlike physical, mechanical and chemical methods
Process is compatible with other pretreatment options
Low energy requirement and low pollution, enzymes are biodegradable and non-toxic agents
Gives potentially high yields of simple sugar (7585% of the theoretical maximum) and improvements are still projected (8595%)
Cell-free enzymes can act in the presence of toxic and recalcitrant substances and under a wide range of conditions (pH, temperature)
Enzymes can work in the presence of inhibitors of microbial metabolism
Absence of acclimatisation period as for the microbial biomass
Easy to control the process
Sludge hydrolysis using enzymes leads to larger volume reduction since when microorganisms are used there is also biomass formation.
Enzymatic hydrolysis is not only energy sparing because of the relatively mild reaction conditions but also avoids the use of toxic and
corrosive chemicals
Low equipment maintenance costs
Disadvantages
The production of commercial enzymes is still costly, although the potential costs are being decreased.
Enzymatic pretreatment demands strict control of reaction conditions.
Enzyme activity is lost through binding to solid matrix, thermal denaturation, active site inactivation, loss of cofactors and reversible and
irreversible inhibition.
Some hydrolytic enzymes such as cellobiase are sensitive to inhibition by their own substrate and end product, cellubiose and glucose.
Biomass degradation is a highly complex multi-enzymatic process, there is still much to learn before enzyme cocktails with increased
activity can efficiently be developed
2011 Informa Healthcare USA, Inc.
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sugars for conversion to biogas. Pilot-scale and fullscale research would facilitate better evaluation of the
technology, its constraints and opportunities. Different
studies in the literature are based on the use of different
types of biomass and a combination of these making
any comparison of the effectiveness of enzyme addition
difficult.
The cost of the enzymes is of prime importance in
order to realise the full potential of enzymatic treatment
of complex biomass. Reducing the cost and improving
the efficiency of separating and converting complex
materials into fermentable sugars is one of the keys to
a viable biogas production from such wastes (Merino
and Cherry, 2007). The enzymes that are presently being
investigated are still expensive because of the cost of their
production. However, this should not thwart the efforts
to carry out more extensive research to identify the most
promising enzymes and determine the optimal conditions for their application. In fact, the results of such
research should provide the incentive for commercial
development to finally produce the enzymes economically at large-scale. The costs are expected to decrease
as technology and techniques advance and as cheaper
growth substrates are explored for cultivating the parent
microorganisms.
The enzymes to be used in hydrolyzing complex wastes
such sludge and agricultural residues do not have to be
highly purified as was demonstrated for lipid-rich wastes.
Such enzyme mixtures may include a variety of enzyme
activities capable of numerous catalytic functions for
them to be useful in heterogeneous substrates like sludge
biomass.
Conclusion
Anaerobic digestion provides biogas, a source of energy
to replace fossil fuels in many parts of the world using
local resources. However, utilization of complex materials such as agricultural crop residues, municipal solid
wastes, market wastes and animal wastes for biogas
production is limited by the hydrolysis step to convert
them into fermentable structures. This review has given
an update of the biotechnological advances towards
improved biogas production using enzymes. During
anaerobic degradation, acidogenic bacteria excrete
hydrolytic enzymes which enable the degradation
of complex organic matter into smaller compounds.
Thus, biological pretreatments using enzymes have
been studied with different materials to intensify biogas production. Pretreatment of lignocellulosic agricultural residues by cellulolytic and hemicellullolytic
enzymes have been reported to yield more biogas.
Similarly, there are several reports of improved biogas
production from lipid-rich waste after pretreatment
with lipases. The use of low-cost enzymatic preparations (lipases, cellulases, proteases) represents a vital
development in the treatment of waste and wastewater
with high organic matter, since the use of high-cost
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Declaration of interest
The authors report no conflicts of interest. The authors
alone are responsible for the content and writing of the
paper.
References
Ahuja SK, Ferreira GM, Moreira AR. (2004). Utilisation of enzymes for
environmental applications. Crit Rev Biotechnol, 24, 125154.
Aitken MD. (1993). Waste treatment applications of enzymes:
opportunities and obstacles. Chem Eng J, 52, B49B58
Akao T, Mizuki E, Saito H, Okumura S, Murao S. (1992). The methane
fermentation of Citrus unshu peel pretreated with fungus enzymes.
Bioresource Technol, 41, 3539.
Amaral AL, Pereira MA, da Motta M, Pons MN, Mota M, Pereira EC,
Alves MM. (2004). Development of image analysis techniques
as a tool to detect and quantify morphological changes in
anaerobic sludge: II. Application to a granule deterioration
process triggered by contact with oleic acid. Biotechnol Bioeng,
87, 194199.
Angelidaki I, Ahring BK. (1992). Effects of free long-chain fatty acids
on thermophilic anaerobic digestion. Appl Microb Biotechnol, 37,
808812.
Angelidaki I, Ahring BK. (2000). Methods of increasing the biogas
potential from the recalcitrant organic matter contained in
manure. Water Sci Technol, 41, 189194.
Attar Y, Mhetre ST, Shawale MD. (1998). Biogas production
enhancement by cellulolytic strains of Actinomycetes. Biogas
Forum, I 72, 1115.
Ayol A. (2005). Enzymatic treatment effects on dewaterability of
anaerobically digested biosolids-I: performance evaluations. Proc
Biochem, 40, 24572434.
Bagi Z, Acs N, Balint KL, Horvath L, Dobo K, Perei KR, Rakhely G,
Kovacs KL. (2007). Biotechnological intensification of biogas
production. Appl Microbiol Biotechnol, 76, 473482.
Bisaria R, Madan M, Mukhopadhyay SN. (1983). Production of biogas
from residues from mushroom cultivation. Biotechnol Lett, 5,
811812.
Bruni E, Jensen AP, Angelidaki I. (2010). Comparative study of
mechanical, hydrothermal, chemical and enzymatic treatments
of digested biofibers to improve biogas production. Bioresour
Technol, 101, 87138717.
Cadoret A, Conrad A, Block J-C. (2002). Availability of low and high
molecular weight substrates to extracellular enzymes in whole
and dispersed activated sludges. Enzyme Microbial Technol, 31,
179186.
Critical Reviews in Biotechnology
14 Parawira Wilson
Mladenovska Z, Ishoy T, Mandiralioglu A, Westermann P, Ahring
BK. (2001). Bioaugmentation of a mesophilic biogas reactor by
anaerobic xylanolytic and cellulolytic bacteria, in Proceedings
of 9th World Congress on Anaerobic Digestion, Part I, ed. by van
Velsen AFM and Verstraete WH, Technologisch Instituut vzw,
Antewerpen, 183188.
Mongkolthanaruk W, Dharmsthiti S. (2002). Biodegradation of lipidrich wastewater by a mixed bacterial consortium. Int Biodeter
Biodegr, 50, 101105.
Mshandete A, Bjornssson L, Kivaisi AK, Rubindamayugi MST,
Mattiasson B. (2007). Enhancement of anaerobic batch digestion
of sisal pulp waste by mesophilic aerobic pre-treatment. Water
Res, 39, 15691575.
Mshandete A, Bjornssson L, Kivaisi AK, Rubindamayugi MST,
Mattiasson B. (2008).Two-stage anaerobic digestion of aerobic
pre-treated sisal leaf decortications residues: hydrolases
activities and biogas production profile. Afr J Biochem Res, 2,
211218.
Muller HW, Trosch W. (1986). Screening of white-rot fungi for
biological pretreatment of wheat straw for biogas production. Appl
Microbiol Biotechnol, 24, 180185.
Noykova N, Muller TG, Gyllenberg M, Timmer J. (2002). Quantitative
analysis of anaerobic wastewater treatment processes:
Identifiability and Parameter Estimation. Biotechnol Bioengin, 78,
89103.
Palatsi J, Laureni M, Andres MV, Flotats X, Nielsen HB, Angelidaki I.
(2009). Strategies for recovering inhibition caused by long chain
fatty acids an anaerobic thermophilic biogas reactors. Bioresour
Technol, 100, 45884596.
Palmisano AC, Schwab BS, Maruseik DA. (1993). Hydrolytic enzyme
activity in landfilled refuse. Appl Microbiol Biotechnol, 38,
828832.
Parawira W. (2004). Anaerobic treatment of agricultural residues and
wastewater: Application of high-rate reactors. PhD thesis, Lund
University, Sweden. ISBN: 9189627-28-8. http://www.lub.lu.se/
luft/diss/tec_848/tec_848_kappa.pdf.
Parawira W, Murto M, Read JS, Mattiasson B. (2005). Profile of
hydrolases and biogas production during two-stage mesophilic
anaerobic digestion of solid potato waste. Process Biochem, 40,
29452952.
Parawira W., and Tekere M. (2011). Biotechnological strategies to
overcome inhibitors in lignocellulose hydrolysates for ethanol
production: review. Crit Rev Biotechnol, 3, 2031.
Parmar N, Singh A, Ward OP. (2001). Enzyme treatment to reduce
solids and improve settling of sewage sludge. J Industrial Microbiol.
Biotechnol, 26, 383386.
Pavlostathis SG, Giraldo-Gomez E. 1991. Kinetics of anaerobic
treatment. Water Sci Technol 24, 3559.
Pereira MA, Cavaleiro AJ, Mota M, Alves MM. (2003). Accumulation of
long-chain fatty acids onto anaerobic sludge under steady state and
shock loading conditions: effect on acetogenic and methanogenic
activity. Water Sci Technol, 48, 3340.
Pereira MA, Souza DZ, Mota M, Alves MM. (2004). Mineralization
of LCFA associated to anaerobic sludge: kinetics, transport
limitations, enhancement of methanogenic activity and effect of
VFA. Biotechnol Bioeng, 88, 502510.
Pereira MA, Pires OC, Mota M, Alves MM. (2005). Anaerobic
biodegradation of oleic and palmitic acids: Evidence of mass
transfer limitations caused by long chain fatty acid accumulation
onto the anaerobic sludge. Biotech Bioeng, 92, 1523.
Perle M, Kimshie S, Shelef G. (1995). Some biochemical aspects of
the anaerobic degradation of dairy wastewater. Water Res, 29,
15491554.
Petruy R, Lettinga G. (1997). Digestion of milk-fat emulsion. Bioresour
Technol, 61, 141149.
Preeti Rao P, Seenayya G. (1994). Improvement of methanogenesis
from cow dung and poultry litter waste digesters by addition of
iron. World J Microbiol Biotechnol, 10, 211214.
Priest FG. (1984). Extracellular enzymes. - Aspects Microbiol, 9, 110.
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